EP2420565B1 - Compositions et procédés utiles pour cultiver des cellules différenciables - Google Patents

Compositions et procédés utiles pour cultiver des cellules différenciables Download PDF

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EP2420565B1
EP2420565B1 EP11174015.5A EP11174015A EP2420565B1 EP 2420565 B1 EP2420565 B1 EP 2420565B1 EP 11174015 A EP11174015 A EP 11174015A EP 2420565 B1 EP2420565 B1 EP 2420565B1
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cells
cell
fgf
culture
insulin
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EP2420565A1 (fr
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Allan Robins
Thomas Schulz
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Viacyte Inc
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Viacyte Inc
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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Definitions

  • the present invention relates to cell culture methods and compositions that are essentially serum-free and comprise a basal salt nutrient solution and an ErbB3 ligand.
  • Human pluripotent cells offer unique opportunities for investigating early stages of human development as well as for therapeutic intervention in several disease states, such as diabetes mellitus and Parkinson's disease.
  • I3-cells derived from human embryonic stem cells hESCs
  • Embryonic stem (ES) cells thus represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can potentially be used to generate an unlimited source of cells suited to transplantation for treatment of diseases that result from cell damage or dysfunction.
  • EPL early primitive ectoderm-like cells as described in International Patent Application WO 99/53021
  • ICM/epiblast in vivo or in vitro derived primitive ectoderm
  • primordial germ cells EG cells
  • EC cells teratocarcinoma cells
  • pluripotent cells derived by dedifferentiation or by nuclear transfer will share some or all of these properties and applications.
  • International Patent Application WO 97/32033 and U.S. Patent No. 5,453,357 describe pluripotent cells including cells from species other than rodents.
  • Human ES cells have been described in International Patent Application WO 00/27995
  • U.S. Patent No. 6,200,806 and human EG cells have been described in International Patent Application WO 98/43679 .
  • hESC based cell replacement therapies will require the development of methods that enable large scale culture and differentiation conditions that are compliant with regulatory guidelines. While several groups have reported simplified growth conditions for hESCs, there are substantial limitations with these studies. To date, however, the successful isolation, long-term clonal maintenance, genetic manipulation and germ line transmission of pluripotent cells has generally been difficult.
  • KSR serum replacer
  • FGF2 has been a component of all these media, it is not clear if it is an absolute necessity, particularly as in some formulations it is necessary to use it at a high concentration (up to 100 ng/ml, Xu et al., 2005 Nature Methods, 2:185-189 ).
  • insulin in their media at ⁇ g/ml levels, or have insulin present due to the use of KSR.
  • Insulin is typically considered to function in glucose metabolism and "cell survival" signaling via binding to the insulin receptor.
  • insulin can also bind to the IGF1 receptor with a lower efficiency and confer classical growth factor activity through the PI3 Kinase/AKT pathway.
  • the presence/requirement for such high levels of insulin ( ⁇ g/ml levels) in KSR or these other media conditions suggests that the major activity is elicited via binding to the IGF1 receptor, which is expressed by hESCs ( Sperger et al., 2003 PNAS, 100(23):13350-13355 ).
  • the EGF growth factor family has at least 14 members, including, but not limited to, EGF, TGF ⁇ , heparin binding-EGF (hb-EGF), neuregulin- ⁇ (also named heregulin- ⁇ (HRG- ⁇ ), glial growth factor and others), HRG- ⁇ , amphiregulin, betacellulin, and epiregulin. All these growth factors contain an EGF domain and are typically first expressed as transmembrane proteins that are processed by metalloproteinase (specifically, ADAM) proteins to generate soluble ectodomain growth factors.
  • ADAM metalloproteinase
  • EGF family members interact with either homo- or heterodimers of the ErbB1, 2, 3 and 4 cell surface receptors with different affinities ( Jones et al., FEBS Lett, 1999, 447:227-231 ).
  • EGF, TGF ⁇ and hbEGF bind ErbB1/1 (EGFR) homodimers and ErbB1/2 heterodimers at high affinity (1-100 nM range), whereas HRG- ⁇ binds ErbB3 and ErbB4 at very high affinity ( ⁇ 1 nM range).
  • Activated ErbB receptors signal through the PI3 Kinase/AKT pathway and also the MAPK pathway.
  • ErbB2 and ErbB3 are amongst the most highly expressed growth factor receptors in hESCs (sperger et al., 2003 PNAS, 100(23):13350-13355 ) and HRG- ⁇ has been shown previously to support the expansion of mouse primordial germ cells ( Toyoda-Ohno et al., 1999 Dev. Biol., 215(2):399-406 ). Furthermore, overexpression and subsequent inappropriate activation of ErbB2 is associated with tumorigenesis ( Neve et al., 2001 Ann. Oncol., 12 Suppl 1:S9-13 ; Zhou & Hung, 2003 Semin.
  • ErbB2 and ErbB3 are expressed in the mouse blastocyst, although not specifically restricted to the inner cell mass (ICM), and ErbB1, EGF and TGF ⁇ are expressed in the human blastocyst ( Chia et al., 1995 Development, 1221(2):299-307 ).
  • HB-EGF has proliferative effects in human IVF blastocyst culture ( Martin et al., 1998 Hum. Reprod., 13(6):1645-52 ; Sargent et al., 1998 Hum. Reprod.
  • Neuregulin-1 is a large gene that exhibits multiple splicing and protein processing variants. This generates a large number of protein isoforms, which are referred to herein collectively as neuregulin. Neuregulin is predominantly expressed as a cell surface transmembrane protein. The extracellular region contains an immunoglobulin-like domain, a carbohydrate modified region and the EGF domain. NRG1 expression isoforms have been reviewed previously ( Falls, 2003 Exp. Cell Res., 284:14-30 ). The cell membrane metalloproteases ADAM 17 and ADAM 19 have been shown to process the transmembrane form(s) of neuregulin-1 to soluble neuregulin/heregulin.
  • HRG- ⁇ , and - ⁇ are the cleaved ectodomains of neuregulin, containing the EGF and other domains.
  • EGF domain is responsible for binding and activation of the ErbB receptors
  • a recombinant molecule containing only this domain can exhibit essentially all of the soluble growth factor effects of this protein ( Jones et al., 1999 FEBS Lett., 447:227-231 ).
  • transmembrane isoforms of neuregulin that are thought to trigger juxtacrine signaling in adjacent cells via interaction of the EGF domain with ErbB receptors.
  • compositions comprising a basal salt nutrient solution and an ErbB3 ligand, with the compositions being essentially free of serum.
  • compositions comprising a basal salt nutrient solution and a means for stimulating ErbB2-directed tyrosine kinase activity in differentiable cells.
  • the invention relates to methods of culturing pluripotent cells, with the methods as defined in the claims.
  • Methods of culturing differentiable cells comprising plating the differentiable cells on a cell culture surface and providing a basal salt nutrient solution to the differentiable cells and a means for stimulating ErbB2-directed tyrosine kinase activity in the differentiable cells.
  • Methods of culturing differentiable cells comprising providing a digest solution to a layer of differentiable cells that are contained in a culture chamber prior to digestion, where the digestion breaks apart the layer of cells into single cells.
  • the single cells are placed into a new tissue culture chamber with a differentiable cell culture solution, wherein the differentiable cell culture solution comprises a basal salt nutrient solution and an ErbB3 ligand.
  • the single differentiable cells are placed in conditions that permit growth and division of the single cells.
  • the term "contacting" i.e., contacting a cell e.g., a differentiable cell, with a compound
  • contacting is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture).
  • the term "contacting” is not intended to include the in vivo exposure of cells to a defined cell medium comprising an ErbB3 ligand, and optionally, a member of the TGF- ⁇ family, that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • the step of contacting the cell with a defined cell medium comprising an ErbB3 ligand, and optionally, a member of the TGF- ⁇ family can be conducted in any suitable manner.
  • the cells may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with the defined medium can be further treated with a cell differentiation environment to stabilize the cells, or to differentiate the cells.
  • differentiate refers to the production of a cell type that is more differentiated than the cell type from which it is derived. The term therefore encompasses cell types that are partially and terminally differentiated.
  • the term "enriched" refers to a cell culture that contains more than approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.
  • the term "effective amount" of a compound refers to that concentration of the compound that is sufficient in the presence of the remaining components of the defined medium to effect the stabilization of the differentiable cell in culture for greater than one month in the absence of a feeder cell and in the absence of serum or serum replacement. This concentration is readily determined by one of ordinary skill in the art.
  • the term "express” refers to the transcription of a polynucleotide or translation of a polypeptide in a cell, such that levels of the molecule are measurably higher in a cell that expresses the molecule than they are in a cell that does not express the molecule.
  • Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.
  • the term “isolated” refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro.
  • the term “isolating” is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.
  • Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, New York ; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York ; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I ; Wu (Ed.) 1979 Meth. Enzymol. 68 ; Wu et al., (Eds.) 1983 Meth. Enzymol.
  • the invention relates to methods comprising a basal salt nutrient solution and an effective amount of an ErbB3 ligand, with the compositions being essentially free of serum.
  • the methods of the present invention are useful for culturing cells, in particular, differentiable cells. It is understood that at different points during culturing the differentiable cells, various components may be added to the cell culture such that the medium can contain components other than those described herein. It is, however, contemplated that at least at one point during the preparation of the culture, or during the culture of the differentiable cells, the defined medium comprises a basal salt nutrient solution and a means for activating ErbB2-directed tyrosine kinase.
  • the term "differentiable cell” is used to describe a cell or population of cells that can differentiate into at least partially mature cells, or that can participate in the differentiation of cells, e.g., fuse with other cells, that can differentiate into at least partially mature cells.
  • “partially mature cells” are cells that exhibit at least one characteristic of the phenotype, such as morphology or protein expression, of a mature cell from the same organ or tissue.
  • a normal, mature hepatocyte typically expresses such proteins as albumin, fibrinogen, alpha-1 -antitrypsin, prothrombin clotting factors, transferrin, and detoxification enzymes such as the cyto chrome P-450s, among others.
  • a “partially mature hepatocyte” may express albumin or another one or more proteins, or begin to take the appearance or function of a normal, mature hepatocyte.
  • a “partially mature pancreatic beta cell” may produce or express the proinsulin protein, among others. The ability of the cells to differentiate into at least partially mature cells will not be dependent upon recombinant engineering techniques, such as transfection, though the cells may, of course, be genetically engineered.
  • the invention contemplates methods useful for differentiable cells, regardless of their source or of their plasticity.
  • the "plasticity" of a cell is used herein roughly as it is in the art. Namely, the plasticity of a cell refers to a cell's ability to differentiate into a particular cell type found in tissues or organs from an embryo, fetus or developed organism. The "more plastic" a cell, the more tissues into which the cell may be able to differentiate.
  • Pluripotent cells include cells and their progeny, which may be able to differentiate into, or give rise to, pluripotent, multipotent, oligopotent and unipotent cells, and/or several, if not all, of the mature or partially mature cell types found in an embryo, fetus or developed organism.
  • Multipotent cells include cells and their progeny, which may be able to differentiate into, or give rise to, multipotent, oligopotent and unipotent progenitor cells, and/or one or more mature or partially mature cell types, except that the mature or partially mature cell types derived from multipotent cells are limited to cells of a particular tissue, organ or organ system.
  • a multipotent hematopoietic progenitor cell and/or its progeny possess the ability to differentiate into or give rise to one or more types of oligopotent cells, such as myeloid progenitor cells and lymphoid progenitor cells, and also give rise to other mature cellular components normally found in the blood.
  • oligopotent cells include cells and their progeny whose ability to differentiate into mature or partially mature cells is more restricted than multipotent cells. Oligopotent cells may, however, still possess the ability to differentiate into oligopotent and unipotent cells, and/or one or more mature or partially mature cell types of a given tissue, organ or organ system.
  • oligopotent cell is a myeloid progenitor cell, which can ultimately give rise to mature or partially mature erythrocytes, platelets, basophils, eosinophils, neutrophils and monocytes.
  • Unipotent cells include cells and their progeny that possess the ability to differentiate or give rise to other unipotent cells and/or one type of mature or partially mature cell type.
  • Differentiable cells may be pluripotent, multipotent, oligopotent or even unipotent.
  • the differentiable cells are pluripotent differentiable cells.
  • the pluripotent differentiable cells are selected from the group consisting of embryonic stem cells, primitive ectoderm cells, primordial germ cells, and teratocarcinoma cells.
  • the differentiable cells are mammalian embryonic stem cells.
  • the differentiable cells are human embryonic stem cells.
  • the invention also contemplates differentiable cells from any source within an animal, provided the cells are differentiable as defined herein.
  • differentiable cells may be harvested from embryos, or any primordial germ layer therein, from placental or chorion tissue, or from more mature tissue such as adult stem cells including, but not limited to adipose, bone marrow, nervous tissue, mammary tissue, liver tissue, pancreas, epithelial, respiratory, gonadal and muscle tissue.
  • the differentiable cells are embryonic stem cells.
  • the differentiable cells are adult stem cells.
  • the stem cells are placental- or chorionic-derived stem cells.
  • the invention contemplates using differentiable cells from any animal capable of generating differentiable cells.
  • the animals from which the differentiable cells are harvested may be vertebrate or invertebrate, mammalian or non-mammalian, human or non-human.
  • animal sources include, but are not limited to, primates, rodents, canines, felines, equines, bovines and porcines.
  • the differentiable cells of the methods of the present invention can be derived using any method known to those of skill in the art.
  • human pluripotent cells can be produced using dedifferentiation.
  • the primitive ectoderm cell used in the present invention can be derived in vivo or in vitro.
  • Primitive ectodermal cells may be generated in adherent culture or as cell aggregates in suspension culture, as described in WO 99/53021 .
  • the human pluripotent cells can be passaged using any method known to those of skill in the art, including, manual passaging methods, and bulk passaging methods such as enzymatic or non-enzymatic passaging.
  • the embryonic stem cells when ES cells are utilized, the embryonic stem cells have a normal karyotype, while in other embodiments, the embryonic stem cells have an abnormal karyotype. In one embodiment, a majority of the embryonic stem cells have a normal karyotype. It is contemplated that greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 95% of metaphases examined will display a normal karyotype.
  • a majority of the embryonic stem cells have an abnormal karyotype. It is contemplated that greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater than 95% of metaphases examined will display an abnormal karyotype. In certain embodiments, the abnormal karyotype is evident after the cells have been cultured for greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 passages. In one specific embodiment, the abnormal karyotype comprises a trisomy of at least one autosomal chromosome, wherein the autosomal chromosome is selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17.
  • the abnormal karyotype comprises a trisomy of more than one autosomal chromosome, wherein at least one of the more than one autosomal chromosomes is selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17.
  • the autosomal chromosome is chromosome 12 or 17.
  • the abnormal karyotype comprises an additional sex chromosome.
  • the karyotype comprises two X chromosomes and one Y chromosome.
  • translocations of chromosomes may occur, and such translocations are encompassed within the term "abnormal karyotype.” Combinations of the foregoing chromosomal abnormalities and other chromosomal abnormalities are also encompassed by the invention.
  • compositions and methods comprise a basal salt nutrient solution.
  • basal salt nutrient solution refers to a mixture of salts that provide cells with water and certain bulk inorganic ions essential for normal cell metabolism, maintain intra- and extra-cellular osmotic balance, provide a carbohydrate as an energy source, and provide a buffering system to maintain the medium within the physiological pH range.
  • basal salt nutrient solutions include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPM1 1640, Ham's F-10, Ham's F-12, ⁇ -Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium, and mixtures thereof.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPM1 1640 Ham's F-10
  • Ham's F-12 Ham's F-12
  • ⁇ MEM ⁇ -Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • Iscove's Modified Dulbecco's Medium and mixtures thereof.
  • the basal salt nutrient solution is an approximately 50:50 mixture of DMEM and Ham's F12.
  • composition can further comprise trace elements.
  • Trace elements can be purchased commercially, for example, from Mediatech.
  • Non-limiting examples of trace elements include but are not limited to compounds comprising, aluminum, chlorine, sulfate, iron, cadmium, cobalt, chromium, germanium, sodium, potassium, calcium, phosphate and magnesium.
  • compounds containing trace elements include but are not limited to, AlCl 3 , AgNO 3 , Ba(C 2 H 3 O 2 ) 2 , CdCl 2 , CdSO 4 , CoCl 2 , CrCl 3 , Cr 2 (SO 4 ) 3 , CuSO 4 , ferric citrate, GeO 2 , KI, KBr, LI, molybdic acid, MnSO 4 , MnCl 2 , NaF, Na 2 SiO 3 NaVO 3 , NH 4 VO 3 , (NH 4 ) 6 Mo 7 O 24 , NiSO 4 , RbCl, selenium, Na 2 SeO 3 , H 2 SeO 3 , selenite ⁇ 2Na, selenomethionone, SnCl 2 , ZnSO 4 , ZrOCl 2 , and mixtures and salts thereof. If selenium, selenite or selenomethionone is present, it is at a concentration of approximately 0.00
  • amino acids can be added to the defined media.
  • Non-limiting examples of such amino acids are Glycine, L-Alanine, L-Alanyl-L-Glutamine, L-Glutamine/Glutamax, L-Arginine hydrochloride, L-Asparagine-H 2 O, L-Aspartic acid, L-Cysteine hydrochloride-H 2 O, L-Cystine 2HCl, L-Glutamic Acid, L-Histidine hydrochloride-H 2 O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt dihydrate, and L-Valine.
  • the amino acid is L-Isoleucine, L-Phenylalanine
  • the defined medium can comprise ascorbic acid.
  • ascorbic acid is present at an initial concentration of approximately 1 mg/L to approximately 1000 mg/L, or from approximately 2 mg/L to approximately 500 mg/L, or from approximately 5 mg/L to approximately 100 mg/L, or from approximately 10 mg/L to approximately 100 mg/L or approximately at 50 mg/L.
  • compositions and methods may also comprise other components such as serum albumin, transferrin, L-glutamine, lipids, antibiotics, 13-Mercaptoethanol, vitamins, minerals, ATP and similar components may be present.
  • vitamins examples include, but are not limited to vitamins A, B1, B2, B3, B5, B6, B7, B9, B12, C, D1, D2, D3, D4, D5, E, tocotrienols, K 1 and K2.
  • concentration of supplements may, for example, be from about 0.001 ⁇ M to about 1mM or more.
  • concentrations at which the supplements may be provided include, but are not limited to about 0.005 ⁇ M, 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 1.0 ⁇ M, 2.0 ⁇ M, 2.5 ⁇ M, 3.0 ⁇ ,M 4.0 ⁇ M, 5.0 ⁇ M, 10 ⁇ M, 20 ⁇ M, 100 ⁇ M, etc.
  • the compositions and methods comprise vitamin B6 and glutamine.
  • the compositions and methods comprise vitamin C and an iron supplement.
  • the compositions and methods comprise vitamin K 1 and vitamin A.
  • the compositions and methods comprise vitamin D3 and ATP.
  • the compositions and methods comprise vitamin B12 and transferrin.
  • compositions and methods comprise tocotrienols and 13-Mercaptoethanol.
  • compositions and methods comprise glutamine and ATP.
  • compositions and methods comprise an omega-3 fatty acid and glutamine.
  • compositions and methods comprise an omega-6 fatty acid and vitamin B 1 .
  • compositions and methods comprise a-linolenic acid and B2.
  • compositions of the present disclosure are essentially serum free.
  • essentially serum free refers to the absence of serum in the solutions. Serum is not an essential ingredient to the methods of the present invention. Thus, the presence of serum in any of the compositions should only be attributable to impurities, e.g., from the starting materials or residual serum from the primary cell culture.
  • essentially serum free medium or environment can contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% serum wherein the presently improved bioactive maintenance capacity of the medium or environment is still observed.
  • the essentially serum free composition does not contain serum or serum replacement, or only contains trace amounts of serum or serum replacement from the isolation of components of the serum or serum replacement that are added to the defined media.
  • the methods of the present invention also comprise a means for stimulating ErbB2 tyrosine kinase activity within differentiable cells.
  • the methods of the present invention comprise the presence of at least one ErbB3 ligand.
  • an ErbB3 ligand will bind the ErbB3 receptor and dimerize with the ErbB2 receptor.
  • the ErbB2 receptor is, in turn, generally responsible for intracellular tyrosine kinase activity within the differentiable cell.
  • ErbB3 ligand refers to a ligand that binds to ErbB3, which in turn dimerizes to ErbB2, thus activating the tyrosine kinase activity of the ErbB2 portion of the ErbB2/ErbB3 heterodimeric receptor.
  • Non-limiting examples of ErbB3 ligands include Neuregulin-1; splice variants and isoforms of Neuregulin-1, including but not limited to HRG-I3, HRG-a, Neu Differentiation Factor (NDF), Acetylcholine Receptor-Inducing Activity (ARIA), Glial Growth Factor 2 (GGF2), and Sensory And Motor Neuron-Derived Factor (SMDF); Neuregulin-2; splice variants and isoforms of Neuregulin-2, including but not limited to NRG2-J3; Epiregulin; and Biregulin.
  • Neuregulin-1 include Neuregulin-1; splice variants and isoforms of Neuregulin-1, including but not limited to HRG-I3, HRG-a, Neu Differentiation Factor (NDF), Acetylcholine Receptor-Inducing Activity (ARIA), Glial Growth Factor 2 (GGF2), and Sensory And Motor Neuron-
  • the means for stimulating ErbB2-directed tyrosine kinase activity comprise at least one ErbB3 ligand that is selected from the group consisting of Neuregulin-1, Heregulin-I3 (HRG-(3), Heregulin-a (HRG-a), Neu differentiation factor (NDF), acetylcholine receptor-inducing activity (ARIA), glial growth factor 2 (GGF2), motor-neuron derived factor (SMDF), Neuregulin-2, Neuregulin-2I3 (NRG2-(3), Epiregulin, Biregulin and variants and functional fragments thereof.
  • the methods of the present invention comprise more than one means for stimulating ErbB2-directed tyrosine kinase activity, such as, but not limited to, using more than one ErbB3 ligand.
  • the ErbB3 ligand is HRG-I3 or a variant or functional fragment thereof.
  • the species from which the culture additive protein, polypeptide or variant or functional fragment thereof derives is the same as the species of cells that are cultured. For example, if mouse ES cells are cultured, an HRG- ⁇ with an amino acid sequence that is identical to the mus musculus HRG- ⁇ sequence can be used as an additive in culture and is considered to be "of the same species.”
  • the species from which the biological additive derives is different from the cells being cultures. For example, if mouse ES cells are cultured, an HRG- ⁇ with an amino acid sequence that is identical to the human HRG-P sequence from can be used as an additive in culture and is considered to be "of different species.”
  • a "functional fragment” is a fragment or splice variant of a full length polypeptide that exerts a similar physiological or cellular effect as the full length polypeptide.
  • the biological effect of the functional fragment need not be identical in scope or strength as the full-length polypeptide, so long as a similar physiological or cellular effect is seen.
  • a functional fragment of HRG- ⁇ can detectably stimulate ErbB2-directed tyrosine kinase.
  • variants include chimeric or fusion polypeptides, homologs, analogs, orthologs, and paralogs.
  • a variant of a reference protein or polypeptide is a protein or polypeptide whose amino acid sequence is at least about 80% identical to the reference protein or polypeptide. In specific embodiments, the variant is at least about 85%, 90%, 95%, 95%, 97%, 98%, 99% or even 100% identical to the reference protein or polypeptide.
  • the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within the reference protein or polypeptide, e.g ., wild-type human or mouse neuregulin-1, and those positions in the modified protein or polypeptide that align with the positions on the reference protein or polypeptide.
  • the amino acid sequence of a subject protein or polypeptide is aligned with the amino acid sequence of a reference protein or polypeptide
  • the sequence that "corresponds to" certain enumerated positions of the reference protein or polypeptide sequence are those that align with these positions of the reference sequence, but are not necessarily in these exact numerical positions of the reference sequence.
  • a polypeptide having an amino acid sequence at least, for example, about 95% "identical" to a reference an amino acid sequence encoding, for example TGF- ⁇ is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence encoding the reference TGF- ⁇ .
  • up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence.
  • These modifications of the reference sequence may occur at the N- terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • identity is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988 ); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993 ); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • identity is well known to skilled artisans ( Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988 )). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J.
  • Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package ( Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984 )), BLASTP, ExPASy, BLASTN, FASTA ( Atschul, S. F., et al., J Molec Biol 215:403 (1990 )) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G.
  • the algorithm used to determine identity between two or more polypeptides is BLASTP.
  • the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference ).
  • FASTDB sequence alignment the query and subject sequences are amino sequences. The result of sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of bases of the query sequence that are N-and C- terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment.
  • the alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the query (subject) sequences or the reference sequence that extend past the N- or C-termini of the reference or subject sequence, respectively, may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue reference sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 reference sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
  • a "chimeric polypeptide” or “fusion polypeptide” comprises at least a portion of a member of the reference polypeptide operatively linked to a second, different polypeptide.
  • the second polypeptide has an amino acid sequence corresponding to a polypeptide which is not substantially identical to the reference polypeptide, and which is derived from the same or a different organism.
  • the term "operatively linked” is intended to indicate that the reference polypeptide and the second polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used.
  • the second polypeptide can be fused to the N-terminus or C-terminus of the reference polypeptide.
  • the fusion polypeptide is a GST-IGF-1 fusion polypeptide in which an IGF-1 sequence is fused to the C-terminus of the GST sequences.
  • Such fusion polypeptides can facilitate the purification of recombinant polypeptides.
  • the fusion polypeptide can contain a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a polypeptide can be increased through use of a heterologous signal sequence.
  • homologs are defined herein as two nucleic acids or polypeptides that have similar, or “identical,” nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists, and antagonists as defined hereafter.
  • the term “homolog” further encompasses nucleic acid molecules that differ from a reference nucleotide sequence due to degeneracy of the genetic code and thus encode the same polypeptide as that encoded by the reference nucleotide sequence.
  • naturally occurring refers to a nucleic or amino acid sequence that occurs in nature.
  • An agonist of a polypeptide can retain substantially the same, or a subset, of the biological activities of the polypeptide.
  • An antagonist of a polypeptide can inhibit one or more of the activities of the naturally occurring form of the polypeptide.
  • the ErbB3 ligand is HRG-I3 or a variant or a functional fragment thereof. Additional, non-limiting examples of ErbB3 ligands are disclosed in United States Patent No. 6,136,558 , 6,387,638 , and 7,063,961 , which are incorporated by reference.
  • Heregulins are generally classified into two major types, alpha and beta, based on two variant EGF-like domains that differ in their C-terminal portions. These EGF-like domains, however, are identical in the spacing of six cysteine residues contained therein. Based on an amino acid sequence comparison, 11040.00tal found that between the first and sixth cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin (AR), 32% identical to TGF-a, and 27% identical to EGF.
  • HB-EGF heparin-binding EGF-like growth factor
  • AR amphiregulin
  • NDF The 44 kDa neu differentiation factor
  • HRG human HRG
  • NDF has an immunoglobulin (Ig) homology domain followed by an EGF-like domain and lacks a N-terminal signal peptide.
  • Ig immunoglobulin
  • pro-NDFs there are at least six distinct fibroblastic pro-NDFs, classified as either alpha or beta polypeptides, based on the sequences of the EGF-like domains.
  • Isoforms 1 to 4 are characterized on the basis of a variable stretch between the EGF-like domain and transmembrane domain.
  • different NDF isoforms are generated by alternative splicing and may perform distinct tissue-specific functions. See EP 505 148 ; WO 93/22424 ; and WO 94/28133 , which are incorporated by reference.
  • the methods are free of exogenous insulin and insulin substitutes.
  • exogenous insulin or insulin substitutes is used herein to indicate insulin or insulin substitutes that is/are not intentionally added to the methods of the present invention.
  • the methods are free of insulin or insulin substitutes that are intentionally supplied.
  • the methods may, however, not necessarily be free of endogenous insulin.
  • endogenous insulin indicates that the cultured cells may be producing insulin of their own accord when cultured according to the methods of the present invention. Endogenous insulin also may be used to indicate residual impurities from the primary cell culture or impurities from the starting materials.
  • the methods of the present invention contain less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ⁇ g/ml of insulin.
  • insulin refers to the protein, or variant or fragment thereof that binds to the insulin receptor in normal physiological concentrations and can induce signaling through the insulin receptor.
  • the term “insulin” encompasses a protein having the polypeptide sequence of native human insulin, or of other mammalian insulin, or of any homologs or variants to these sequences. Additionally, the temi insulin encompasses polypeptide fragments that are capable of binding to the insulin receptor to induce signaling through the insulin receptor.
  • insulin substitute refers to any zinc containing compound that may be used in place of insulin to give substantially similar results as insulin. Examples of insulin substitutes include, but are not limited to zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate.
  • insulin-like growth factors are not insulin substitutes or homologs of insulin, as contemplated in the present invention.
  • the methods of the present invention comprise the use of at least one insulin-like growth factor (IGF) or a variant or a functional fragment thereof.
  • the methods of the present invention are free of any exogenous insulin-like growth factors (IGFs).
  • the methods of the present invention contain less than 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml of IGF-1.
  • the term "activator of IGF-1R” refers to mitogens that play a pivotal role in regulating cell proliferation, differentiation, and apoptosis.
  • the effects of an activator of IGF-1R are typically mediated through IGF-1R, although they can be mediated through other receptors.
  • the IGF-1R is also involved in cell transformation induced by tumor virus proteins and oncogene products, and the interaction is regulated by a group of specific binding proteins (IGFBPs).
  • IGFBPs specific binding proteins
  • a large group of IGFBP proteases hydrolyze IGFBPs, resulting in the release of bound IGFs that then resume their ability to interact with IGF-IR.
  • the ligands, the receptors, the binding proteins, and the proteases are all considered to be activators of IGF-1R.
  • the activator of IGF-1R is IGF-1, or IGF-2.
  • the activators of IGF-1R is an IGF-1 analog.
  • IGF-1 analogs include LongR3-IGF1, Des(1-3)IGF-1, [ArgIIGF-1, [Ala 31 ]IFG-1, Des(2,3)[Ala 31 ]IGF-1, [Leu 24 ]IGF1, Des(2,3)[Leu 24 ]IGF-1, [Leu 60 ]IGF-1, [Ala 31 ][Leu 60 ]IGF-1, [Leu 24 ][Ala 31 ]IGF-1, and combinations thereof.
  • the IFG-1 analog is LongR3-IGF1, which is a recombinant analog of human insulin growth factor-1.
  • LongR3-IGF1 is initially present at a concentration of approximately 1 ng/ml to approximately 1000 ng/ml, more preferably approximately 5 ng/ml to approximately 500 ng/ml, more preferably approximately 50 ng/ml to approximately 500 ng/ml, more preferably approximately 100 ng/ml to approximately 300 ng/ml, or at a concentration of approximately 100 ng/ml.
  • the methods of the present invention comprise transforming growth factor beta (TGF-(3) or a TGF-I3 family member or variants or functional fragments thereof.
  • TGF-(3) transforming growth factor beta
  • TGF-I3 family member or variants or functional fragments thereof the term "member of the TGF-I3 family” or the like refers to growth factors that are generally characterized by one of skill in the art as belonging to the TGF-13 family, either due to homology with known members of the TGF-I3 family, or due to similarity in function with known members of the TGF-I3 family.
  • the TGF-I3 family member of variant or functional fragment thereof activates SMAD 2 or 3.
  • the member of the TGF-I3 family is selected from the group consisting of Nodal, Activin A, Activin B, TGF-13, bone morphogenic protein-2 (BMP2) and bone morphogenic protein-4 (BMP4).
  • the member of the TGF-13 family is Activin A.
  • Nodal it is initially present at a concentration of approximately 0.1 ng/ml to approximately 2000 ng/ml, more preferably approximately 1 ng/ml to approximately 1000 ng/ml, more preferably approximately 10 ng/ml to approximately 750 ng/ml, or more preferably approximately 25 ng/ml to approximately 500 ng/ml.
  • Activin A is initially present at a concentration of approximately 0.01 ng/ml to approximately 1000 ng/ml, more preferably approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably approximately 0.1 ng/ml to approximately 25 ng/ml, or most preferably at a concentration of approximately 10 ng/ml. It is contemplated that if present, TGF-13 is initially present at a concentration of approximately 0.01 ng/ml to approximately 100 ng/ml, more preferably approximately 0.1 ng/ml to approximately 50 ng/ml, or more preferably approximately 0.1 ng/ml to approximately 20 ng/ml.
  • the methods of the present invention are free of activators of FGF receptors.
  • activators of FGF receptors There are currently at least 22 known members of the family of fibroblast growth factors, with these factors binding to one of at least one of four FGF receptors.
  • the term "activator of an FGF receptor" refers to growth factors that are generally characterized by one of skill in the art as belonging to the FGF family, either due to homology with known members of the FGF family, or due to similarity in function with known members of the FGF family.
  • the activator of an FGF receptor is an FGF, such as, but not limited to a-FGF and FGF2.
  • the compositions and methods are free of exogenous FGF2.
  • exogenous FGF2 is used herein to indicate fibroblast growth factor 2, i.e., basic FGF, that is not intentionally added to the methods of the present invention.
  • the methods are free of intentionally supplied FGF2.
  • the methods may, however, not necessarily be free of endogenous FGF2.
  • endogenous FGF2 indicates that the cultured cells may be producing FGF2 of their own accord when cultured according to the methods of the present invention.
  • Endogenous FGF2 also may be used to indicate residual impurities from the primary cell culture or impurities from the starting materials.
  • the compositions and methods of the present contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml of FGF2.
  • the methods of the invention can include at least one activator of an FGF receptor, including any of the FGF polypeptides, functional fragments thereof or variants thereof. It is contemplated that if FGF2 is present, it is initially present at a concentration of approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably approximately 0.5 ng/ml to approximately 50 ng/ml, more preferably approximately 1 ng/ml to approximately 25 ng/ml, more preferably approximately 1 ng/ml to approximately 12 ng/ml, or most preferably at a concentration of approximately 8 ng/ml. In another specific embodiment, the methods of the invention can include at least one activator of an FGF receptor, other than FGF2.
  • the methods of the present invention may comprise at least one of FGF-7, FGF-10 or FGF-22 or variants or functional fragments thereof.
  • FGF-7, FGF-10 and FGF-22, or variants or functional fragments thereof are present.
  • FGF-7, FGF-10 or FGF-22 or variants or functional fragments are present, each is initially present at a concentration of approximately 0.1 ng/ml to approximately 100 ng/ml, more specifically from approximately 0.5 ng/ml to approximately 50 ng/ml, more specifically from approximately 1 ng/ml to approximately 25 ng/ml, more specifically from approximately 1 ng/ml to approximately 12 ng/ml, or most specifically at a concentration of approximately 8 ng/ml.
  • the methods of the present invention comprise serum albumin (SA).
  • SA is either bovine SA (BSA) or human SA (HAS).
  • BSA bovine SA
  • HAS human SA
  • concentration of the SA is more than about 0.2%, volume to volume (v/v), but less than about 10% v/v.
  • the concentration of SA is more than about 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6% and 9.8% (v/v).
  • compositions and methods comprise at least one insoluble substrate.
  • the differentiable cells may be placed on a cell culture surface that comprises such compounds as, but is not limited to, polystyrene, polypropylene.
  • the surface may, in turn, be coated with an insoluble substrate.
  • the insoluble substrate is selected from the group consisting of a collagen, a fibronectin and fragments or variants thereof
  • Other examples of insoluble substrates include, but are not limited to, fibrin, elastin, fibronectins, laminins and nidogens.
  • the cell culture environments and methods of the present invention comprise plating the cells in an adherent culture.
  • the terms “plated” and “plating” refer to any process that allows a cell to be grown in adherent culture.
  • adherent culture refers to a cell culture system whereby cells are cultured on a solid surface, which may in turn be coated with an insoluble substrate that may in turn be coated with another surface coat of a substrate, such as those listed below, or any other chemical or biological material that allows the cells to proliferate or be stabilized in culture.
  • the cells may or may not tightly adhere to the solid surface or to the substrate.
  • the substrate for the adherent culture may comprise any one or combination of polyornithine, laminin, poly-lysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycans, poly glycolytic acid (PGA), poly lactic acid (PLA), and poly lactic-glycolic acid (PLGA).
  • the substrate for the adherent culture may comprise the matrix laid down by a feeder layer, or laid down by the pluripotent human cell or cell culture.
  • extracellular matrix encompasses solid substrates such as but not limited to those described above, as well as the matrix laid down by a feeder cell layer or by the pluripotent human cell or cell culture.
  • the cells are plated on MATRIGELTM-coated plates.
  • the cells are plated on fibronectin-coated plates.
  • the plates are prepared by coating with 10 ⁇ g/ml human plasma fibronectin (Invitrogen, #33016-015), diluted in tissue grade water, for 2-3 hours at room temperature.
  • serum can be placed in the medium for up to 24 hours to allow cells to plate to the plastic. If using serum to promote the attachment of the cells, the media is then removed and the compositions, which are essentially serum-free, are added to the plated cells.
  • a "feeder cell” is a cell that grows in vitro, that is co-cultured with a target cell and stabilizes the target cell in its current state of differentiation.
  • a "feeder cell layer” can be used interchangeably with the term “feeder cell.”
  • the term "essentially free of a feeder cell” refers to tissue culture conditions that do not contain feeder cells, or that contain a de minimus number of feeder cells. By “de minimus”, it is meant that number of feeder cells that are carried over to the instant culture conditions from previous culture conditions where the differentiable cells may have been cultured on feeder cells.
  • conditioned medium is obtained from a feeder cell that stabilizes the target cell in its current state of differentiation.
  • the defined medium is a non-conditioned medium, which is a medium that is not obtained from a feeder cell.
  • the term "stabilize,” when used in reference to the differentiation state of a cell or culture of cells, indicates that the cells will continue to proliferate over multiple passages in culture, and preferably indefinitely in culture, where most, if not all, of the cells in the culture are of the same differentiation state.
  • the stabilized cells divide, the division typically yield cells of the same cell type or yield cells of the same differentiation state.
  • a stabilized cell or cell population in general does not further differentiate or de-differentiate if the cell culture conditions are not altered, and the cells continue to be passaged and are not overgrown.
  • the cell that is stabilized is capable of proliferation in the stable state indefinitely, or for at least more than 2 passages.
  • the cells are stable for more than 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, more than 10 passages, more than 15 passages, more than 20 passages, more than 25 passages, or more than 30 passages.
  • the cell is stable for greater than approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months of continuous passaging.
  • the cell is stable for greater than approximately 1 year of continuous passaging.
  • stem cells are maintained in culture in a pluripotent state by routine passage in the defined medium until it is desired that they be differentiated.
  • proliferate refers to an increase in the number cells in a cell culture.
  • compositions and methods comprise an inactivator of BMP signaling.
  • an "inactivator of BMP signaling” refers to an agent that antagonizes the activity of one or more BMP proteins or any of their upstream or downstream signaling components through any of its possible signaling pathways.
  • the compound(s) used to inactivate BMP signaling can be any compound known in the art, or later discovered.
  • Non-limiting examples of inactivators of BMP signaling include dominant-negative, truncated BMP receptor, soluble BMP receptors, BMP receptor-Fc chimeras, noggin, follistatin, chordin, gremlin, cerberus/DAN family proteins, ventropin, high dose activin, and amnionless.
  • compositions and methods can comprise at least one hormone, cytokine, adipokine, growth hormone or variant or functional fragment thereof.
  • the growth hormone present in the defined medium will be of the same species as the differentiable cells that are cultured with the defined media.
  • the growth hormone is human growth hormone.
  • the use of growth hormone that is from a species different than the cultured cells is also contemplated.
  • the hormone, cytokine, adipokine and/or growth hormone is present at an initial concentration of approximately 0.001 ng/ml to approximately 1000 ng/ml, more preferably approximately 0.001 ng/ml to approximately 250 ng/ml, or more preferably approximately 0.01 ng/ml to approximately 150 ng/ml.
  • cytokines and adipokines examples include, but are not limited to, the four a-helix bundle family of cytokines, the interleukin -1 (IL-1) family of cytokines, the IL-17 family of cytokines and the chemokine family of cytokines.
  • IL-1 interleukin -1
  • the invention contemplates members and subclasses of each of these families of cytokines, such as, but not limited to, the CC chemokines, the CXC chemokines, the C chemokines and the CX 3 C chemokines, interferons, interleukins, lymphotoxins, c-kit ligand, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte-macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), leptin, adiponectin, resistin, plasminogen activator inhibitor-1 (PAI- 1), tumor necrosis factor-alpha (TNFa), tumor necrosis factor-beta (TNF(3), leukemia inhibitory factor, visfatin, retinol binding protein 4 (RBP4), erythropoietin (EPO), thrombopoietin (THPO
  • the present invention relates to methods of culturing differentiable cells, with the methods comprising plating differentiable cells on a cell culture surface, providing a basal salt nutrient solution to the cells and providing a means for stimulating ErbB2-directed tyrosine kinase activity in the cells.
  • differentiable cells are contacted with the at least one of the compositions disclosed herein in the absence of serum or serum replacement, and in the absence of a feeder cell layer, such that the cells are maintained in an undifferentiated state for at least one month.
  • Pluripotency can be determined through characterization of the cells with respect to surface markers, transcriptional markers, karyotype, and ability to differentiate to cells of the three germ layers. These characteristics are well known to those of ordinary skill in the art.
  • the differentiable cells can be passaged using enzymatic, non-enzymatic, or manual dissociation methods prior to and/or after contact with the defined medium of the invention.
  • enzymatic dissociation methods include the use of proteases such as trypsin, collagenase, dispase, and ACCUTASETM.
  • ACCUTASETM is used to passage the contacted cells.
  • the resultant culture can comprise a mixture of singlets, doublets, triplets, and clumps of cells that vary in size depending on the enzyme used.
  • a non-limiting example of a non-enzymatic dissociation method is a cell dispersal buffer.
  • methods of culturing differentiable cells comprise providing a dissociation solution to a layer of stem cells that are contained in a culture chamber prior to dissociation, where the dissociation breaks apart the layer of cells into single cells.
  • the single cells are placed into a new tissue culture chamber with a stem cell culture solution, wherein the stem cell culture solution comprises a basal salt nutrient solution and an ErbB3 ligand. Once cultured, the single stem cells are placed in conditions that permit growth and division of the single cells.
  • the disaggregation solution used in the methods of the present invention can be any disaggregation solution capable of breaking apart or disaggregating the cells into single cells, without causing extensive toxicity to the cells.
  • disaggregation solutions include, but are not limited to, trypsin, ACCUTASETM, 0.25% Trypsin/EDTA, TrypLE, or VERSENETM (EDTA) and trypsin.
  • the methods of the present invention need not result in every cell of the confluent layer being disaggregated into single cells, provided that at least a few single cells are disaggregated and capable of being re-cultured.
  • Differentiable cells may also be utilized to screen for molecules or factors that influence their plasticity or other characterisitics.
  • differentiable cells could be used to identify agents that induce apoptosis, differentiation or proliferation, as well as similar effects in differentiated lineages that have been generated from the differentiable cells.
  • FIG. 16 shows the morphology and alkaline phosphatase staining of BG02 cells that were cultured in DC-HALF in both a 96-well and 384-well plate, using the methods described herein. Briefly, hESCs cells that were split, using ACCUTASETM, and plated in 96-well and 384-well plates and cultured showed a similar plating efficiency as what is observed using other culture dishes. In addition, the cells formed colonies, and were expanded successfully over 5 days in the smaller environments.
  • hESCs could also be grown in 96-well culture devices (not shown) that provide real-time measurements of impedance, which can be used to measure cell proliferation and viability using the RT-CESTm methods from ACEA Biosciences, Inc. ( www.aceabio.com ). Such an approach would enable a label-free identification and quantitation of subtle or immediate effects on differentiable cells, as well as measurements of proliferation, apoptosis and changes to morphology, in real time.
  • the methods of the invention may contain virtually any combination of the components set out above or described elsewhere herein, provided the compositions and methods comprise a basal salt nutrient solution and a means for stimulating ErbB2 directed tyrosine kinase activity.
  • the components of the methods of the invention will vary according to the protocol design. Accordingly, one embodiment of the present invention relates to culturing differentiable cells in 96-well plates and/or 384-well plates. Indeed, using the methods of the present invention, the cell culture chamber, i.e., the culture dish, is no longer limited to specific dimensions. Thus, the methods of the present invention is not limited to specific culture chamber dimensions.
  • compositions and methods described herein have several useful features.
  • the compositions and methods described herein are useful for modeling the early stages of human development.
  • the compositions and methods described herein can also serve for therapeutic intervention in disease states, such as neurodegenerative disorders, diabetes mellitus or renal failure, such as by the development of pure tissue or cell type.
  • the cell types that differentiate from differentiable cells have several uses in various fields of research and development including but not limited to drug discovery, drug development and testing, toxicology, production of cells for therapeutic purposes as well as basic science research. These cell types express molecules that are of interest in a wide range of research fields. These include the molecules known to be required for the functioning of the various cell types as described in standard reference texts. These molecules include, but are not limited to, cytokines, growth factors, cytokine receptors, extracellular matrix, transcription factors, secreted polypeptides and other molecules, and growth factor receptors.
  • the differentiable cells of the invention can be differentiated through contact with a cell differentiation environment.
  • the term "cell differentiation environment” refers to a cell culture condition wherein the differentiable cells are induced to differentiate, or are induced to become a human cell culture enriched in differentiated cells.
  • the differentiated cell lineage induced by the growth factor will be homogeneous in nature.
  • the term “homogeneous,” refers to a population that contains more than approximately 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired cell lineage.
  • a cell differentiating medium or environment may be utilized to partially, terminally, or reversibly differentiate the differentiable cells of the present invention.
  • the medium of the cell differentiation environment may contain a variety of components including, for example, KODMEM medium (Knockout Dulbecco's Modified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetal bovine serum), FGF2 (fibroblast growth factor 2), KSR or hLIF (human leukemia inhibitory factor).
  • the cell differentiation environment can also contain supplements such as L-Glutamine, NEAA (non-essential amino acids), P/S (penicillin/streptomycin), N2, B27 and ⁇ -mercaptoethanol ( ⁇ -ME).
  • additional factors may be added to the cell differentiation environment, including, but not limited to, fibronectin, laminin, heparin, heparin sulfate, retinoic acid, members of the epidermal growth factor family (EGFs), members of the fibroblast growth factor family (FGFs) including FGF2 and/or FGF8, members of the platelet derived growth factor family (PDGFs), transforming growth factor (TGF)/ bone morphogenetic protein (BMP)/ growth and differentiation factor (GDF) factor family antagonists including but not limited to noggin, follistatin, chordin, gremlin, cerberus/DAN family proteins, ventropin, high dose activin, and amnionless or variants or functional fragments thereof.
  • EGFs epidermal growth factor family
  • FGFs fibroblast growth factor family
  • PDGFs platelet derived growth factor family
  • TGF transforming growth factor
  • BMP bone morphogenetic protein
  • GDF growth and differentiation factor
  • TGF/BMP/GDF antagonists could also be added in the form of TGF/BMP/GDF receptor-Fc chimeras.
  • Other factors that may be added include molecules that can activate or inactivate signaling through Notch receptor family, including but not limited to proteins of the Delta-like and Jagged families as well as inhibitors of Notch processing or cleavage, or variants or functional fragments thereof.
  • growth factors may include members of the insulin like growth factor family (IGF), insulin, the wingless related (WNT) factor family, and the hedgehog factor family or variants or functional fragments thereof Additional factors may be added to promote mesendoderm stem/progenitor, endoderm stem/progenitor, mesoderm stem/progenitor, or definitive endoderm stem/progenitor proliferation and survival as well as survival and differentiation of derivatives of these progenitors.
  • IGF insulin like growth factor family
  • WNT wingless related
  • Hhog factor family or variants or functional fragments thereof Additional factors may be added to promote mesendoderm stem/progenitor, endoderm stem/progenitor, mesoderm stem/progenitor, or definitive endoderm stem/progenitor proliferation and survival as well as survival and differentiation of derivatives of these progenitors.
  • compositions described herein are useful for the screening of test compounds to determine whether a test compound modulates pluripotency, proliferation, and/or differentiation of differentiable cells.
  • Pluripotency, proliferation and/or differentiation of differentiable cells can be readily ascertained by one of ordinary skill in the art. Non-limiting methods include examining cell morphology, the expression of various markers, teratoma formation, cell counts and measurements of impedance.
  • the progression of the differentiable cells to the desired cell lineage, or its maintenance in an undifferentiated state can be monitored by quantitating expression of marker genes characteristic of the desired cell lineage as well as the lack of expression of marker genes characteristic of differentiable cell types.
  • One method of quantitating gene expression of such marker genes is through the use of quantitative PCR (Q-PCR). Methods of performing Q-PCR are well known in the art. Other methods that are known in the art can also be used to quantitate marker gene expression. Marker gene expression can be detected by using antibodies specific for the marker gene of interest.
  • the screening method encompasses methods of identifying a compound capable of modulating pluripotency, proliferation and/or differentiation of a differentiable cell, comprising (a) providing a differentiable cell; (b) culturing the cell in a composition comprising a basal salt nutrient solution and an ErbB3 ligand, wherein the composition is essentially serum free; (c) contacting the cell with a test compound; and determining whether an increase or decrease in pluripotency, proliferation and/or differentiation occurs in the cell contacted with the compound, said increase being an indication that the compound modulates pluripotency, proliferation and/or differentiation.
  • the ErbB3 ligand is HRG- ⁇ .
  • the ErbB3 ligand can be substituted with a test compound, to determine the effects of the test compound.
  • the effects on pluripotency, proliferation and/or differentiation that occurs with the test compound can be compared to the effects on pluripotency, proliferation and/or differentiation that occurs with the ErbB3 ligand to determine the effects of the test compound on the differentiable cells. It is contemplated that any of the compositions described herein can be used in the screening methods of the present invention.
  • the cells can be cultured in the absence of an ErbB3 ligand (ErbB2-directed tyrosine kinase activity) to determine the effects of the absence of an ErbB3 ligand (ErbB2-directed tyrosine kinase activity) on the cells.
  • an ErbB3 ligand ErbB2-directed tyrosine kinase activity
  • compositions comprising the desired cell lineage that are substantially free of other cell types can be produced.
  • compositions comprising mixtures of the differentiable cells and the desired cell lineage can also be produced.
  • cells of the desired cell lineage can be isolated by using an affinity tag that is specific for such cells.
  • an affinity tag specific for a target cell is an antibody that is specific to a marker polypeptide that is present on the cell surface of the target cell but which is not substantially present on other cell types that would be found in a cell culture produced by the methods described herein.
  • kits wherein the kit comprises a basal salt nutrient solution and at least one compound capable of stimulating ErbB2-directed tyrosine kinase activity.
  • the kits may comprise at least one ErbB3 ligand, as described herein.
  • the kits may comprise more than one ErbB3 ligand.
  • the kits may comprise at least one of TGF-I3 or a TGF-I3 family member or a variant or functional fragment thereof as described herein.
  • the kits may comprise more than one of TGF-I3 or a TGF-I3 family member or a variant or functional fragment thereof.
  • the kits may comprise at least one fibroblast growth factor or variant or functional fragment thereof.
  • the kits may comprise more than one fibroblast growth factor or variant or functional fragment thereof.
  • kits may comprise at least one of FGF-7, FGF-1 0, FGF-22 or variants or functional fragments thereof.
  • the kits may comprise serum albumin.
  • the kits may comprise serum and/or at least one insoluble substrate as described herein and/or at least one disaggregation solution.
  • kits may contain virtually any combination of the components set out above or described elsewhere herein. As one skilled in the art would recognize, the components supplied with kits of the invention will vary with the intended use for the kits. Thus, kits may be designed to perform various functions set out in this application and the components of such kits will vary accordingly.
  • the human embryonic stem cell line BGO1v (BresaGen, Inc., Athens, GA) was used in some of the experiments described herein.
  • the BGO1v hESC line is a karyotypically variant cell line, which exhibits stable karyotype containing specific trisomies (karyotype: 49, XXY,+12,+17).
  • Parent cultures were maintained as described previously ( Schulz et al., 2003, BMC Neurosci., 4:27 ; Schulz et al., 2004, Stem Cells, 22(7):1218-38 ; Rosler et al., 2004, Dev.
  • the cells were grown in dishes coated with MATRIGELTM or fibronectin, in conditioned media from mouse embryonic fibroblasts (MEFs) (MEF-CM) comprising DMEM:F12 with 20% KSR, 8 ng/ml FGF2, 2 mM L-Glutamine, lx non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM13-mercaptoethanol (Sigma, St. Louis, Missouri, USA), with collagenase passaging.
  • MEFs mouse embryonic fibroblasts
  • the defined culture (DC) media tested herein comprised DMEM/F12, 2 mM Glutamax, lx non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 10 ⁇ g/ml transferrin (all from Invitrogen, Carlsbad, California, USA) 0.1 mM13-mercaptoethanol (Sigma), 0.2% fatty acid-free Cohn's fraction V BSA (Serologicals), lx Trace Element mixes A, B and C (Cellgro) and 50 ⁇ g/ml Ascorbic Acid (Sigma).
  • Variable levels of recombinant growth factors were used, including FGF2 (Sigma), LongR3-IGF1 (JRH Biosciences), Heregulin-I3 EGF domain (HRGI3, Peprotech), TGFI3 (R&D systems), nodal (R&D systems), LIF (R&D systems), EGF (R&D systems), TGFa (R&D systems), HRGa (R&D systems), BMP4 (R&D systems), and Activin A (R&D Systems).
  • LongR3-IGFl is a modified version of IGF1 that has reduced affinity for IGF1 binding proteins, some of which are expressed in hESCs.
  • DC-HALF is the defined culture media as above, containing 10 ng/ml HRG-I3, 10 ng/ml Activin A, 200 ng/ml LR-IGF1 and 8 ng/ml FGF2.
  • DC-HAI is defined culture media as above containing 10 ng/ml HRG- ⁇ , 10 ng/ml Activin A, and 200 ng/ml LR-IGF1.
  • the LR-IGF1 component can, of course be replaced with IFG1.
  • MATRIGELTM coated dishes were prepared by diluting Growth Factor Reduced BD MATRIGELTM matrix (BD Biosciences, Franklin Lakes, New Jersey, USA) to a final concentration range of about 1:30 to about 1:1000 in cold DMEM/F-12. In one embodiment, the concentration of MATRIGELTM is about 1:200. 1 ml/35 mm dish was used to coat dishes for 1-2 hours at room temperature or at least overnight at 4°C. Plates were stored up to one week at 4°C. MATRIGELTM solution was removed immediately before use.
  • parent cultures were plated into 6-well dishes for comparison of multiple conditions. Cultures were typically plated directly into the test conditions. The cultures were assessed every day and graded based on morphological criteria 4 to 5 days after plating. The grading scale of 1 to 5 involved examining the whole culture and assessing overall proportion of undifferentiated colonies, their relative size, and proportion of colonies or parts of colonies exhibiting obvious differentiation. Grade 5 indicates "ideal" cultures, with large undifferentiated colonies and negligible differentiation. Grade 4 indicates a very good culture, but with some obvious differentiation. Grade 3 indicates an acceptable culture, but with around half the colonies exhibiting obvious differentiation. Grade 2 cultures are predominantly differentiated, with occasional putative undifferentiated cells. Grade 1 cultures contain differentiated colonies or the cultures did not adhere or did not survive. Cultures that exhibited good expansion of undifferentiated cells were passaged to assess longer-term culture in these conditions.
  • BG01v cells cultured in DC media as described above containing 100 ng/ml LongR3-IGF1 (LR-IGF1), 8 ng/ml FGF2 and 1 ng/ml Activin A were harvested and RNA was prepared using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. First strand cDNA was prepared using the iScript kit (Biorad) and real time PCR was carried out using a MJ Research Opticon thermal cycler.
  • the EGF domain family of ligands bind to the ErbB family of receptor tyrosine kinases.
  • BG01v cells were plated in 6 well trays on MATRIGELTM diluted at 1:1000, in defined culture medium (DC) containing 100 ng/ml LongR3-IGF1, 8 ng/ml FGF2 and 1 ng/ml Activin A.
  • DC defined culture medium
  • DMSO carrier control
  • 50 nM-20 ⁇ M AG1478 an ErbB1 inhibitor
  • 100 nM-20 ⁇ M AG879 an ErbB2 inhibitor
  • Reference example 3 - BG01v cells are Maintained in Defined Media Containing Heregulin
  • BGOlv cells have active endogenous ErbB signaling and that they may also respond to exogenous HRG- ⁇ .
  • BG01v cells were grown in DC medium containing 10 ng/ml HRG- ⁇ , 200 ng/ml LongR3-IGF1, 8 ng/ml FGF2 and 10 ng/ml Activin A, on MATRIGELTM diluted 1:1000 ( Figures 3A and B ). These cells were grown for 4 passages, or >20 days, exhibited undifferentiated morphology and did not show elevated spontaneous differentiation.
  • BG01v cells were also maintained for 2 passages, or >13 days, in DC medium comprising 10 ng/ml HRG ⁇ , 200 ng/ml LongR3-IGF1, and 10 ng/ml Activin A. These cultures proliferated normally and exhibited very low spontaneous differentiation, demonstrating that BG01v cells could be maintained in defined conditions with HRG ⁇ in the absence of FGF2.
  • RT-PCR demonstrated that mESCs express ADAM19, Neuregulin1 (Nrg1), and ErbB1-4 ( Figures 4A ).
  • Nrg1 Neuregulin1
  • ErbB1-4 Figures 4A .
  • ErbB2 and 3 appeared to be expressed at higher levels than ErbB1, with low levels of ErbB4 being detected.
  • MEFs mouse embryonic fibroblasts
  • Figure 4B The expression of the ErbB receptor transcripts in mouse embryonic fibroblasts (MEFs) was also examined ( Figure 4B ).
  • MEFs are a heterogenous population of cells derived from E12.5-13.5 viscera that have been used historically to maintain mouse and human EC cells and ES cells. Expression ofNrgl and Adam19 in this population suggests that the HRG- ⁇ ectodomain is also present in MEF-conditioned media and may exert significant effects upon pluripotency.
  • DMSO and 1-50 ⁇ M AG1478 had no apparent affect on cell proliferation, with subconfluent colonies of alkaline phosphatase positive mESCs observed ( Figures 5A-C ). However, AG879 substantially inhibited cell growth at 50 ⁇ M (compare Figures 5D and 5F ) and may have slowed proliferation at 20 ⁇ M ( Figure 5E ). mESCs grown in AG879 did not appear to differentiate and maintained a pluripotent morphology, and alkaline phosphatase activity.
  • tyrphostin AG825 Another highly selective inhibitor of the ErbB2 tyrosine kinase, tyrphostin AG825 ( Murillo, et al. 2001 Cancer Res 61, 7408-7412 ), was used to investigate the role of ErbB2 in human ESCs.
  • AG825 significantly inhibited proliferation of hESCs growing in conditioned medium (CM) ( Fig. 6A ).
  • CM conditioned medium
  • AG825 inhibited proliferation without widespread cell death, and viable hESCs could be maintained for >5 days (not shown).
  • Western blotting showed that AG825 inhibited autophosphorylation of ErbB2 at tyrosine-1248 in starved/heregulin (HRG) pulsed hESCs growing in DC-HAIF ( Fig. 6B ).
  • a quantitative assay was performed by plating 2x10 5 cells/well in 6-well trays on 1:1000 MATRIGELTM, in selected combinations of 10 or 50 ng/ml HRG- ⁇ , 10 ng/ml EGF, 1000 U/ml LIF or 10 ng/ml BMP4. The cultures were grown for 8 days, fixed, and the number of alkaline phosphatase colonies was counted ( Figure 7A ). No colonies were observed in defined conditions without growth factors, and ⁇ 45 colonies were observed with HRG-I3, HRG-I3/EGF and HRG-I3/BMP combinations.
  • BG02 cells cultured in DC-HALF for 6 months (25 passages) maintained the potential to form complex teratomas ( Fig. 8A ) and representatives of the three germ layers in vitro ( Fig. 8B ).
  • Transcriptional analyses were used to compare global expression in hESCs cells ( Liu et al 2006, BMC Dev Biol 6, 20 ) maintained in CM and DC-HALF. Greater than 11,600 transcripts were detected in BG02 cells grown in DC-HALF for 10 and 32 passages, and BG02 cells grown in CM for 64 passages. There were about 10364 transcripts common to all populations ( Fig.
  • compositions of the present disclosure can be used as a simple medium for supporting self-renewal of differentiable cells.
  • DC-HAIF cultures were initially expanded on culture dished coated with growth factor-reduced MATRIGEL TM1:30, but could also be maintained successfully long-term on this substrate diluted 1:200 ( Fig. 9A ), or 1:1000.
  • BG02 and CyT49 hESCs could also be maintained for >5 passages on tissue culture dishes coated with human serum ( Fig. 9B ); human fibronectin ( Fig. 9C ); or VITROGROTM ( Fig. 9D ), which is a proprietary humanized ECM.
  • Fluorescence in situ hybridization analysis demonstrated that the cells cultured and passaged in DC-HAIF maintained expected copy numbers for hChr12 (98% 2-copy), hChr17 (98% 2-copy), hChrX (95% 1-copy) and hChrY (98% 1-copy) ( Fig. 12C ).
  • Karyotyping analysis also demonstrated that a normal euploid chromosome content and banding profile was maintained in these cells
  • hESCs Essentially all of the reported culture conditions for hESCs to date include supraphysiological levels of insulin, which can stimulate both IR and IGF1R. To distinguish the activities that insulin and insulin-substitutes exert, compared to IGF1, hESCs are cultured in defined media conditions in physiological levels of these growth factors. The concentrations of insulin and IGF1 are titrated from about 0.2 to about 200 ng/ml and cell proliferation is monitored by counting cells after 5 days. Cultures that expand successfully are serially passaged 5 times.
  • IGF1 Physiological levels of IGF1 support the expansion of hESC cultures, whereas physiological levels of insulin do not, indicating that the activity of insulin or insulin-substitutes cannot replace IGF1, and that IGF1 and insulin (or insulin substitutes) represent separate classes of biological activities with regard to action on hESCs.
  • BG02 cells are split using ACCUTASETM and 1 x 10 5 cells/well are plated in 6-well trays in defined culture (DC) media.
  • the DC media contains 10 ng/ml HRG- ⁇ , 200 ng/ml LongR3-IGF1, and 10 ng/ml FGF10.
  • Vitamin B 6 (0.5 ⁇ M) and/or Vitamin B 12 (0.5 ⁇ M) are added to experimental wells. Cell numbers in each condition are counted after 7 days. Cell counting and colony counting of both experimental and control wells will provide insight on the effects of Vitamin B 6 and Vitamin B 12 on cell growth.
  • markers of differentiation such as OCT4 can be assayed in the experimental well to determine the effects of the additives and supplements to the differentiation state of the differentiable cells.
  • BG02 cells were maintained long term in DC-HAI, for 20 passages ( Fig. 13A ), and BG01 cells were also serially passaged in DC-HAI, both in the absence of FGF2. The cultures did not deteriorate or exhibit overt differentiation, and exhibited normal expansion of undifferentiated colonies throughout the culture period. The maintenance of a normal male karyotype in a BG02 culture was demonstrated after 6 passages in DC-HAI ( Fig. 13B , 20/20 normal metaphase spreads).
  • Arrays were scanned with an Illumina Bead Array Reader confocal scanner and primary data processing, background subtraction, and data analysis were performed using Illumina BeadStudio software according to the manufacturer's instructions. A minimum detection confidence score of 0.99 (a computed cutoff indicating the target sequence signal was distinguishable from the negative controls) was used to discriminate the presence or absence of transcript expression. Data analysis was performed using parallel approaches described for other hESC samples ( Liu et al 2006, BMC Dev Biol 6:20 ).
  • BG02 cells maintained in DC-HAI differentiated to representatives of mesoderm, endoderm and ectoderm in complex teratomas formed in SCID-beige mice (not shown), formally demonstrating the maintenance ofpluripotency in cultures grown in the absence of exogenous FGF2.
  • BG01 cells were passaged with ACCUTASETM and grown in defined conditions containing only 10 ng/ml HRG- ⁇ and 200 ng/ml LongR3-IGF1 (DC-HI). These DC-HI cultures were maintained for 10 passages, and did not exhibit overt differentiation or a slowing of proliferation.

Claims (14)

  1. Méthode pour la culture et le maintien de cellules pluripotentes dans un état pluripotent stabilisé, comprenant le dépôt des cellules pluripotentes sur une surface de culture de cellules, la fourniture d'une solution nutritive saline basale aux cellules pluripotentes et la fourniture d'un moyen pour la stimulation de l'activité de tyrosine kinase dirigée vers ErbB2 dans les cellules pluripotentes, dans laquelle le moyen pour la stimulation de l'activité de tyrosine kinase dirigée vers ErbB2 comprend un ligand qui se lie spécifiquement à ErbB3.
  2. Méthode selon la revendication 1, dans laquelle il n'est fourni ni insuline, ni substitut d'insuline auxdites cellules.
  3. Méthode selon l'une quelconque des revendications précédentes, comprenant en outre la fourniture d'un facteur de croissance semblable à l'insuline ou d'un fragment fonctionnel de celui-ci.
  4. Méthode selon l'une quelconque des revendications 1 à 3, dans laquelle ledit ligand de ErbB3 est choisi dans le groupe constitué de Neuréguline-1, Héréguline-β (HRG-β), Héréguline-ct (HRG-ct), facteur de différenciation Neu (NDF), activité d'induction du récepteur de l'acétylcholine (ARIA), facteur de croissance glial 2 (GGF2), facteur dérivé de motoneurone (SMDF), Neuréguline-2, Neuréguline-2β (NRG2-β), Epiréguline, Biréguline et de fragments fonctionnels de ceux-ci.
  5. Méthode selon l'une quelconque des revendications précédentes, comprenant en outre la fourniture d'un facteur de croissance transformant bêta (TGFβ), d'un membre de la famille des TGFβ ou d'un fragment fonctionnel de ceux-ci.
  6. Méthode selon la revendication 5, dans laquelle ledit membre de la famille des TGFβ est choisi dans le groupe constitué de Nodal, Activine A, Activine B, protéine de morphogénèse de l'os 2 (BMP2), protéine de morphogénèse de l'os 4 (BMP4) et de fragments fonctionnels de ceux-ci.
  7. Méthode selon l'une quelconque des revendications précédentes, dans laquelle il n'est pas fourni de facteur de croissance des fibroblastes exogène auxdites cellules.
  8. Méthode selon l'une quelconque des revendications 1 à 6, comprenant en outre la fourniture d'au moins un facteur de croissance des fibroblastes (FGF) choisi dans le groupe constitué de FGF-2, FGF-7, FGF-10, FGF-22 et de fragments fonctionnels variants de ceux-ci.
  9. Méthode selon l'une quelconque des revendications précédentes, comprenant en outre la fourniture d'une albumine de sérum (SA).
  10. Méthode selon la revendication 9, dans laquelle la SA est une SA bovine (BSA) ou une SA humaine (HSA).
  11. Méthode selon la revendication 9 ou 10, dans laquelle la concentration de la SA est de plus d'environ 0,2%, volume sur volume (v/v).
  12. Méthode selon l'une quelconque des revendications 9 à 11, dans laquelle la concentration de la SA est de moins d'environ 5% v/v.
  13. Méthode selon l'une quelconque des revendications précédentes, dans laquelle les cellules pluripotentes sont des cellules souches pluripotentes.
  14. Méthode selon la revendication 1, dans laquelle les cellules pluripotentes sont des cellules souches embryonnaires de primate.
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US8211699B2 (en) 2012-07-03
US20140154802A1 (en) 2014-06-05
CN105802904B (zh) 2021-04-20
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EP2420565A1 (fr) 2012-02-22
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JP5719919B2 (ja) 2015-05-20
EP1994141A4 (fr) 2009-04-22
US8153429B2 (en) 2012-04-10
CA2643478A1 (fr) 2007-09-07
US20080113433A1 (en) 2008-05-15
CN105802904A (zh) 2016-07-27
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JP2016005462A (ja) 2016-01-14
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JP5830217B2 (ja) 2015-12-09
SG170021A1 (en) 2011-04-29
WO2007101130A3 (fr) 2007-12-27
AU2007220759A1 (en) 2007-09-07
DK1994141T3 (en) 2018-02-12
EP1994141A2 (fr) 2008-11-26
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JP2014097063A (ja) 2014-05-29
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US20130309708A1 (en) 2013-11-21
US8658352B2 (en) 2014-02-25
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